The conservation of number and location of 18S sites indicates the relative stability of rDNA in species of Pentatomomorpha (Heteroptera)

Fluorescent in situ hybridization (FISH) with rDNA probes has been used for comparative cytogenetics studies in different groups of organisms. Although heteropterans are a large suborder within Hemiptera, studies using rDNA are limited to the infraorder Cimicomorpha, in which rDNA sites are present in the autosomes or sex chromosomes. We isolated and sequenced a conserved 18S rDNA region of Antiteuchus tripterus (Pentatomidae) and used it as a probe against chromosomes of 25 species belonging to five different families of Pentatomomorpha. The clone pAt05, with a length of 736 bp, exhibited a conserved stretch of 590 bp. FISH analysis with the probe pAt05 always demonstrated hybridization signals in sub-terminal positions, except for Euschistus heros. Apparently, there is a tendency for 18S rDNA sites to locate in autosomes, except for Leptoglossus gonagra and Euryophthalmus rufipennis, which showed signals in the m-and sex chromosomes, respectively. Although FISH has produced evidence that rearrangements are involved in rDNA repositioning, whether in different autosomes or between sex and m-chromosomes, we have no conclusive evidence of what were the pathways of these rearrangements based on the evolutionary history of the species studied here. Nevertheless, the diversity in the number of species analyzed here showed a tendency of 18S rDNA to remain among the autosomes.Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES

Origin and distribution of AT-rich repetitive DNA families in Triatoma infestans (Heteroptera)

Triatoma infestans, one of the most important vectors of Trypanosoma cruzi, is very interesting model, because it shows large interpopulation variation in the amount and distribution of heterochromatin. This polymorphism involved the three large pairs up to almost all autosomal pairs, including the sex chromosomes. To understand the dynamics of heterochromatin variation in T. infestans, we isolated the AT-rich satDNA portion of this insect using reassociation kinetics (C(0)t), followed by cloning, sequencing and FISH. After chromosome localization, immunolabeling with anti-5-methylcytosine, anti-H4K5ac and anti-H3K9me2 antibodies was performed to determine the functional characteristics of heterochromatin. The results allowed us to reorganize the karyotype of T. infestans in accordance with the distribution of the families of repetitive DNA using seven different markers. We found that two arrays with lengths of 79 and 33 bp have a strong relationship with transposable element sequences, suggesting that these two families of satDNA probably originated from Polintons. The results also allowed us to identify at least four chromosome rearrangements involved in the amplification/dispersion of AT-rich satDNA of T. infestans. These data should be very useful in new studies including those examining the cytogenomic and population aspects of this very important species of insect. (C) 2014 Elsevier B.V. All rights reserved.Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES

Distribution of 45S and 5S rDNA sites in 23 species of Eleocharis (Cyperaceae)

Studies of rDNA location in holocentric chromosomes of the Cyperaceae are scarce, but a few reports have indicated the occurrence of multiple 45S rDNA sites at terminal positions, and in the decondensed state of these regions in prometaphase/metaphase. To extend our knowledge of the number 45S and 5S rDNA sites and distribution in holocentric chromosomes of the Cyperaceae, 23 Brazilian species of Eleocharis were studied. FISH showed 45S rDNA signals always located in terminal regions, which varied from two (E. bonariensis with 2n = 20) to ten (E. flavescens with 2n = 10 and E. laeviglumis with 2n = 60). 5S rDNA showed less variation, with 16 species exhibiting two sites and 7 species four sites, preferentially at terminal positions, except for four species (E. subarticulata, E. flavescens, E. sellowiana and E. geniculata) that showed interstitial sites. The results are discussed in order to understand the predominance of terminal rDNA sites, the mechanisms involved in the interstitial positioning of 5S rDNA sites in some species, and the events of amplification and dispersion of 45S rDNA terminal sites.Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq

Karyotypic diversification and its contribution to the taxonomy of Eleocharis (Cyperaceae) from Brazil

A karyotype analysis of 147 populations of 25 Brazilian species of Eleocharis (Cyperaceae) was carried out, including representatives of the three subgenera that occur in the country: Limnochloa, Scirpidium and Eleocharis. The analyses showed chromosomes without centromeres, but with terminal nucleolar constrictions (satellites) in some chromosomes. The chromosome numbers varied from 2n = 6 in E. subarticulata and E. maculosa to 2n = 60 in E. laeviglumis, but the chromosome basic number x = 5 was confirmed. Species of the subgenera Eleocharis and Scirpidium possess fewer and larger chromosomes, while those in the subgenus Limnochloa have small and more numerous chromosomes. These features indicate that the karyotypes of the subgenera Eleocharis and Scirpidium are more closely related, in agreement with morphological and phylogenetical data. The representatives of the section Eleocharis exhibited the largest differences in chromosome number and size, probably due to chromosome fission and fusion. Polyploidy was the most common event in this group. Nevertheless, most of the studied species exhibited regular meiosis with only bivalent formation, even the polyploids, such as in E. geniculata and E. sellowiana. The cytogenetic information obtained showed quite variable karyotypes with chromosomes gradually decreasing in size, and predominance of polyploidy. These results are useful in the differentiation of the subgenera.Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq

Developmental programmed cell death during asymmetric microsporogenesis in holocentric species of Rhynchospora (Cyperaceae)

International audienceMembers of the Cyperaceae family exhibit an asymmetric microsporogenesis that results in the degeneration of three out of four meiotic products. Efforts have been made previously to describe the resulting structure, named the pseudomonad, but mechanisms concerning the establishment of cell domains, nuclear development, and programmed cell death are largely unknown. Using the Rhynchospora genus as a model, evidence for cell asymmetry, cytoplasmic isolation, and programmed cell death was obtained by a combination of electron microscopic, cytochemical, immunocytochemical, in situ hybridization, and flow cytometric methods. Degenerative cells were identified at the abaxial region, with the cytoskeleton marking their delimitation from the functional domain after meiosis. After attempting to initiate cell division with an unreplicated genome and abnormal spindle assembly, these cells exhibited a gradual process of cytoplasmic contraction associated with hypermethylation of cytosines and differential loss of DNA. These results indicate that the asymmetric tetrad establishes a functional cell, where one nucleus is preferentially selected to survive. Degenerative haploid cells are then eliminated in a multistep process associated with mitotic disorder, non-random elimination of repetitive DNA, vacuolar cell death, and DNA fragmentation

FISH using a gag-like fragment probe reveals a common Ty3-gypsy-like retrotransposon in genome of Coffea species

The genus Coffea possesses about 100 species, and the most economically important are Coffea canephora and Coffea arabica. The latter is predominantly self-compatible with 2n = 4x = 44, while the others of the genus are diploid with 2n = 2x = 22 and mostly self-incompatible. Studies using molecular markers have been useful to detect differences between genomes in Coffea; however, molecular and cytogenetic studies have produced only limited information on the karyotypes organization. We used DOP-PCR to isolate repetitive elements from genome of Coffea arabica var. typica. The pCa06 clone, containing a fragment of 775 bp length, was characterized by sequencing and used as a probe in chromosomes of C. arabica and six other species: C. canephora, Coffea eugenioides, Coffea kapakata, Coffea liberica var. dewevrei, Coffea racemosa, and Coffea stenophylla. This insert shows similarities with a gag protein of the Ty3-gypsy-like super-family. Dot blot and FISH analyses demonstrated that pCa06 is differentially accumulated between species and chromosomes. Signals appeared scattered and clustered on the chromosomes and were also associated with heterochromatic regions. While the literature shows that there is a high karyotype similarity between Coffea species, our results point out differences in the accumulation and dispersion of this Ty3-gypsy-like retrotransposon during karyotype differentiation of Coffea.Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq

Cytogenetic characterization reveals differences in nuclear organization of cystic cells among Brazilian species of Triatoma (Heteroptera, Reduviidae)

Cytogenetic studies in triatomines have described the occurrence of holokinetic chromosomes, heterochromatin distribution and the location of rDNA (ribosomal DNA) sites, but few aspects of nuclear organization in this group have been discussed. We have focused on ultrastructural and cytogenetic features and differences in cystic cells of seminiferous tubules between five species of Triatoma. Cystic cells showed evidence of polyploidy events and heterochromatic blocks appeared predominantly in the central region of the nuclei. Cytogenetic analyses showed that there was variation in chromocenter number between species, and that the central regions were AT-rich [DAPI(+) (4',6-diamidino-2-phenylindole(+))], whereas the periphery was CG-rich (CMA(+)). Another characteristic was the distribution of 45S rDNA, which differed according to the chromosomal location of this sequence. In all we have compared aspects of nuclear organization, polyploidy, heterochromatin, rDNA site distribution and methylation levels, as well as the relationships between five species of Triatoma from a cystic cell perspective.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES